Allogeneic hematopoietic cell transplantation (HCT) is an effective therapy for life-threatening, non-malignant disorders of the hematopoietic and immune systems, such as marrow failure syndromes, hemoglobinopathies, immunodeficiency disorders and lysosomal storage diseases. Major limitations of allogeneic HCT in patients with nonmalignant disorders have been host-versus-graft reactions (graft rejection) and immune reactions of donor lymphocytes against host antigens, also called graft-versus-host disease (GVHD), both of which can be fatal. To circumvent the problems of graft rejection and GVHD, allogeneic HCT recipients are generally treated with combinations of immunosuppressive agents for extended periods of time. Long term immunosuppression, however, also weakens host immune responses to pathogens, thereby increasing the risk of serious infections - and is not uniformly successful in controlling GVHD. Therefore, efforts at promoting immune tolerance without compromising immune competence are needed to improve outcomes in HCT. CD28 and CTLA-4 are leukocyte cell-surface costimulatory receptors that profoundly influence the course of immune responses: CD28 magnifies the effects of TCR signaling and enhances both cell cycle progression and T cell survival; CTLA-4 (CD152) provides opposing inhibitory signals. CD28 and CTLA-4 bind the shared, related ligands B7.1 (CD80) and B7.2 (CD86). The long-term goal of this project is to develop rationally engineered antagonists and agonists specific for CD28 or CTLA-4 for use as short-term immunotherapeutics in various clinical contexts, initially focusing on the control of host-versus-graft reactions and GVHD following allogeneic HCT. Our hypothesis is that administering a CD28 antagonist along with a CTLA-4 agonist shortly after transplantation should lead to down-regulation of both host and donor T cells that were specifically activated in response to mismatched major and minor antigens, prevent recruitment and induce apoptosis in newly activated T cells. Ideal reagents would be maximally selective for CD28 or CTLA-4, have short biological half-lives to reduce immune related adverse events and minimal molecular weights to maximize tissue penetrance. The immediate goal of the exploratory/developmental phase of this project (this R21 application) is to computationally redesign soluble forms of B7.1 and B7.2 into receptor-specific binding reagents, confirming their properties biochemically, that would next be evaluated in in vitro cell-based assays and in vivo studies (in the canine model of histocompatible marrow transplantation) in subsequent collaborative applications. The small size of these reagents optimizes tissue penetrance and short biological half-life; we predict that monomeric forms will act as competitive antagonists and that multimeric (dimeric Fc fusions or tetravalent reagents) will act as agonists. This approach leverages an iterative protein engineering pipeline we successfully established for the development of computationally-designed AIDS vaccine immunogens (epitope-scaffolds) for a distinct therapeutic application based on novel B7 redesign targets.